1. Modulation of Microphthalmia-associated Transcription Factor Gene Expression Alters Skin Pigmentation 
C.B. Lin, L. Babiarz, F. Liebel, M. Kizoulis, G.J. Gendimenico, M. Seiberg, Dr 
Journal of Investigative Dermatology 
Volume 119, Issue 6, Pages (December 2002)
DOI: /j x
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Modulation of MITF promoter activity. HaCaT, B16, and melan-a cells, transfected with the MITF promoter-luciferase construct (pGL2.hMIP) were treated, at 48 h after transfection, for 7 h with (a) forskolin (100 μM), UVB (30 mJ per cm2) and the combination of forskolin and UVB. (b) Transfected B16 cells were treated with 30 mJ per cm2 for the times indicated. (c) Transfected B16 cells were treated with increasing doses of UVB for 7 h. (d) Transfected B16 cells were treated with increasing concentrations of TCM-1 and combination of TCM-1 (0.1 mg per ml) and forskolin (100 μM) as indicated. Measured luciferase activity is expressed as percent change of MITF promoter activity relative to untreated control (mean±SEM).
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
DHLA, LA, and resveratrol inhibit forskolin and UVB-induced MITF promoter activity. B16 cells transfected with the MITF promoter-luciferase construct (pGL2.hMIP) were treated 48 h after transfection, with (a) Increasing concentrations of DHLA, LA, and resveratrol. (b) Forskolin alone (100 μM), or in combination with DHLA (300 and 400 μM), LA (400 μM), or resveratrol (20 and 30 μg per ml); (c) UVB alone (30 mJ per cm2), or in combination with DHLA (200 and 400 μM). Measured luciferase activity is expressed as percent increase of MITF promoter activity relative to untreated control (mean±SEM). (d) B16 cells were treated with TCM-1, DHLA, forskolin, and cell permeable 8-bromo-cAMP at the concentrations indicated for 7 h. Measured cellular cAMP is expressed as percent increase of cAMP concentration relative to untreated control (mean±SEM).
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
DHLA inhibits tyrosinase and TRP-1 promoter activities via the inhibition of MITF promoter. (a) DHLA inhibits MITF, tyrosinase, and TRP-1 promoter activities. Melan-a and B16 cells were transfected with MITF, tyrosinase, or TRP-1 promoter luciferase reporter constructs. At 48 h after transfection, cells were treated with forskolin (100 μM), DHLA (400 μM), or their combination for 7 h. Measured luciferase activity is expressed as percent of the basal luciferase activity relative to untreated control (mean±SEM). (b,c) MITF rescues DHLA and LA-inhibited tyrosinase activity. B16 cells were transfected with either MITF or tyrosinase promoter luciferase reporter constructs and with either vector (pEBB) or MITF expression plasmid (pEBB.bMi). Cell lysates were prepared following 24 h incubation with LA or DHLA. MITF overexpression rescues the tyrosinase promoter activity inhibited by LA or DHLA (b), but has no effect on MITF transcription (c). (d) Modulation of endogenous MITF mRNA levels. NHEM were treated with DHLA (350 μM), LA (350 μM), and resveratrol (20 μg per ml) for 1 or 2 d, and with forskolin (100 μM) or TCM-1 (0.1 mg per ml) for 2 h, 1 d, or 2 d. Total RNA (20 μg) was analyzed by northern blots, probed for MITF expression. The relative intensity of the MITF mRNA band is expressed as percent of MITF signal intensity relative to untreated control, normalized to the 28S rRNA ethidium bromide stain. (e) Modulation of endogenous MITF and tyrosinase protein levels. Protein extracts from B16 cells treated with DHLA (350 μM), LA (350 μM), and forskolin (100 μM) for indicated times, were western blotted and probed with anti-Mi antibodies followed by anti-tubulin and with αPEP-7 (anti-tyrosinase) antibodies.
Journal of Investigative Dermatology  , DOI: ( /j x)
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5. Figure 4
DHLA, LA, and TCM-1 treatments affect tyrosinase activity. (a) Tyrosinase activity analysis of B16 cells, treated with test compounds. B16 cells treated for 24 h with forskolin (100 μM), DHLA (400 μM), LA (400 μM), and TCM-1 (0.1 mg per ml) were lyzed and incubated with 100 μl of 0.1% DOPA for 3 h. Tyrosinase activity was determined by measuring absorbance at 490 nm. Data expressed as percent change of the tyrosinase activity relative to untreated control (mean±SEM). (b) Analysis of tyrosinase enzymatic activity in DHLA-treated cells and lysates. B16 cells were either lyzed and then incubated with DHLA and DOPA for 3 h (open bars) or treated with DHLA in culture and then lyzed (hatched bars). The effect of DHLA on the enzymatic activity of tyrosinase in the lysate (open bars) was compared with the effect of DHLA on live cells (hatched bars). (c–k) Modulation of tyrosinase activity in situ. NHEM were treated with 100 μM forskolin (d), 0.1 mg TCM-1 per ml (e), 350 μM DHLA (f), 350 μM LA (g), 20 μg resveratrol per ml (h), forskolin and DHLA (i), forskolin and LA (j), and TCM-1 and DHLA (k). Untreated control is shown in (c). Cells were DOPA stained following 48 h incubation with test agents.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
DHLA reduces skin pigmentation in vivo. Dark-skinned Yucatan swine were topically treated with 1% LA, 1% resveratrol per ml, 1% DHLA or vehicle, twice a day, 5 d per wk, for 8 wk. (a) Picture of a treated site, taken at 8 wk of treatment, demonstrating DHLA-induced skin lightening. (b,c) F&M staining of histologic sections of untreated (b) and DHLA-treated (c) sites. (d) Relative pigmentation (melanin area divided by total epidermis area), as analyzed using computerized imaging.
Journal of Investigative Dermatology  , DOI: ( /j x)
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7. Figure 6
Prevention of UVB-induced tanning. Light-skinned Yucatan swine were UVB treated with 1 mean erythema dose to create visible tanning as described in Materials and Methods, with or without daily treatment of test material. Tanning was observed in UVB alone and in UVB and vehicle-treated sites, and was reduced or prevented with LA, DHLA, and resveratrol treatments. F&M staining of histologic sections confirm this observation: (a) untreated control; (b) UVB plus vehicle; (c) UVB plus LA (1%); (d) UVB plus DHLA (1%); (e) UVB plus resveratrol (1%). Relative pigmentation (melanin area divided by total epidermis area), as analyzed using computerized imaging, is shown in (f).
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions